Nucleosides and nucleotides, components of nucleic acids, and oligonucleotides (short stretches of nucleic acids) find broad application among others, in biology, medicine and nanotechnology (Agrawal, 1996; Cotter, 1996; Crooke, 1995; Ells, 1996; Jefferies and De ClercQ, 1995; Keller and Manak, 1993; Niemeyer, 1997; Seeman et al, 1998; Wiedbrauk and Farkas, 1995).
Due to the different requirements dependent upon specific applications of oligonucleotides and their components, natural nucleosides, nucleotides and oligonucleotides are modified in order to obtain molecules with designed and needed properties. Essentially all parts of the nucleoside, nucleotide and oligonucleotide can be modified, namely the sugar residue, nucleic base and phosphate group (Agrawal, 1993; Micklefield, 2001; Shabarova and Bogdanov, 1994; Sanghvi and Cook, 1994; Uhlmann, E., Peyman, A., 1990).
One of the modifications is based on the use of boron clusters such as carboranes, as the modifying entity. The carborane group can be attached to the nucleoside unit directly or through a linker. Most nucleosides modified with a carborane group belong to a pyrimidine nucleoside family. (Lesnikowski i Schinazi, 1995; Tjarks, W., 2000).
Three types of carboranyl group (—C2B10H12) containing DNA-oligonucleotides have been described so far: 1) (o-carboran-1-yl)methylphosphonate-oligonucleotides (CBMP-oligonucleotides), consisting of a carborane cage within an internucleotide linkage (Lesnikowski and Schinazi, 1993; Lesnikowski et al, 1997; Lesnikowski, 2003), 2) 5-(o-carboran-1-yl)-2′-deoxyuridine-oligonucleotides (CDU-oligonucleotides) containing a carborane cage attached to a nucleobase (Fulcrand-El Kattan et al, 1994; Lesnikowski et al, 1996; Lesnikowski, 2003), and 3) 2′-O-(o-carboran-1-yl)methyl-oligonucleotides (2′-CBM-oligonucleotides) with a carborane cage linked to a sugar residue at 2′ position (Olejniczak et al, 2002; Olejniczak and Lesnikowski, 2002; Lesnikowski, 2003). Other types of modified nucleosides, nucleotides and oligonucleotides are derivatives of these compounds consisting of metal complexes (Bigey et al., 1997; Dervan et al., 1988; Dougan et al., 1997; Dubey et al., 2000; Hurley and Tor, 1998; Ossipov et al, 2001; Strobel et al, 1988; Yu et al, 2000). Nucleoside conjugates containing metal complexes with ligands other than a carborane cage or nucleosides containing a carborane cage without a metal ion have been described. For example, U.S. Pat. No. 6,180,766 discloses nucleosides and oligonucleotides containing boron clusters. Application of these derivatives as boron carriers for BNCT of tumors, antisense biotherapeutics and molecular probes in medical diagnostics are proposed.
Application WO 2002053571 describes new derivatives of polycyclic hydrocarbons and naphthalene imides type of ferrocene and methods of their synthesis, their intercalating properties, method for their electrochemical detection and several examples of practical applications. The disclosed compounds are easy for synthesis and purification, and are efficient intercalators towards double stranded DNA and RNA. However, as an electrochemical label it binds to a nucleic acid molecule through weak intercalating interactions, substantially limiting the practical utility of the proposed compounds.
The subject of JP 2001064298 is the preparation of oligonucleotide/ferrocene conjugates and their application for electrochemical detection of DNA. Disclosed is a method for synthesis of conductively or electrochemically active oligonucleotides. Oligonucleotides are transformed into an organic solvent soluble form via ion pair formation with suitable lipids and are treated with a type of ferrocene monocarboxylic acid N-hydroxy succinimide ester compound. Though, in the invention described above, the method for the synthesis of oligonucleotide conjugates containing a metal ion is limited to the incorporation into the oligonucleotide molecule iron only.
In the U.S. Pat. No. 5,599,796 the method and compounds for treating urogenital tumors, and particular, cancer of the prostate, bladder, and kidney with BCNT, are disclosed. Preferred boron carriers include 5-carboranyl-2′-deoxyuridine (CDU) and 5-o-carboranyl-1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil (CFAU). Nucleosides and oligonucleotides bearing an —O-[(carboran-1-yl)alkyl]phosphate, S-[(carboran-1-yl)alkyl]phosphorothioate, or Se-[(carboran-1-yl)alkyl]phosphoroselenoate in place of the (carboran-1-yl)phosphonate moiety can be used.
Oligonucleotides of specific gene sequences that include one or more 3′,5′-linking-(carboran-1-yl)phosphonate moieties can also be used in antisense therapy in the selective modification of gene expression. The therapy is accomplished by administering the boron-containing compound by any appropriate route, including by intravenous injection, oral delivery or by catheter or other direct means, in such a manner that the compound accumulates in the target tumor. After desired accumulation of the compound in the tumor, the site is irradiated with an effective amount of low energy neutrons.
In the application JP 2002080491 preparation of electrophilic boron-containing nucleotide analogs and their intermediates is disclosed. Described nucleotide anlogues of general structure (HO)2BX[P(O)(O—)O]nNu (1; Nu=nucleoside residue; X=CF2, CH2; n=2, 3) are prepared by difluoromethylation of HP(O)(OR1)2 (R1=alkyl), reaction with (R2O)3B (R2=lower alkyl), deprotection, protection with 1,2-diols, coupling with nucleotides, and deprotection.
Application U.S. Pat. No. 5,130,302 is focused on boronated nucleoside, nucleotide and oligonucleotide compounds, compositions and methods for using the same. A novel class of pharmaceutically active boronated nucleosides are provided. The nucleosides are boronated at a nitrogen of the purine or pyrimidine ring or analogues thereof. Also provided are phosphate esters of these nucleosides and oligomers thereof. Methods of making and use of the boronated nucleosides are also disclosed. Both of the above applications provide method for the synthesis of some boron containing nucleoside derivatives, but does not allow incorporation of more than one boron atom per modification and does not permit incorporation into a modified molecule other than boron metals.
U.S. Pat. No. 5,466,679 discloses carboranyl uridines and their use in BNCT. The invention relates to novel boron-containing nucleosides and amino acids which can utilize the enzymatic systems in tumor cells for incorporating such boron-containing structures into nucleic acids and proteins. Subsequent use of boron neutron capture therapy provides a method for treatment of tumor cells. The subject of U.S. Pat. No. 5,171,849 is 2′ and 3′ carboranyl uridines and their diethyl ether adducts. Disclosed is also a process for preparing carboranyl uridine nucleoside compounds and their diethyl ether adducts which exhibit a tenfold increase in boron content over prior art boron containing nucleoside compounds. Said carboranyl uridine nucleoside compounds exhibit enhanced lipophilicity and hydrophilic properties adequate to enable solvation in aqueous media for subsequent incorporation of said compounds in methods for boron neutron capture therapy in mammalian tumor cells. In the U.S. Pat. No. 5,405,598 new sensitizing agents for use in boron neutron capture therapy are proposed. In spite that the compound proposed in the last three applications contain tenfold more boron atoms than described earlier derivatives consisting boric acid residue, the compounds still contain twofold less than proposed in the present invention. It should be also pointed out that any of the methods proposed so far do not allow incorporation into designed biomolecule such as nucleoside, nucleic acid or other broad spectrum of different metal ions.
Nucleic acid hybridization technology essentially began with the work of Hall and Spiegelman (Hall and Spiegelman, 1961). Originally, the probe and target were hybridized in a solution and the hybrids were isolated by equilibrium-density gradient centrifugation. This procedure was slow, labor-intensive and inaccurate. Substantial progress has been achieved due to the development of the first solid phase hybridization method (Denhardt, 1966). Extensive studies during next 20 years yielded new, automated techniques for hybridization technology. The advancement was possible mainly due to improved attachment methods of DNA/RNA or DNA-oligonucleotide probes to solid support and better methods of their labeling and detection (Keller and Manak, 1993). The most frequently used labels were radioactive isotopes such as 32P, 3H, 14C and 35S, fluorescent compounds and dyes absorbing visible light, and the most often used test format was the ELISA type immunoenzymatic assay. Comparison of selected methods are shown in Table 1.
TABLE 1Comparison of sensitivity of selected methods used for DNA detection.EnzymeLabelMethod of detectionSensitivityAlkaline4-nitrophenylColorimetric5fmolphosphateAlkalineNBT/BCIPColorimetric0.5fmolphosphateHorse-radisho-PhenylenediamineColorimetric0.1fmolperoxidaseHorse-radishLuminolColorimetric1.0fmolperoxidaseHorse-radishENH/LUMChemiluminescence0.05fmolperoxidase—32PScintillation0.05fmol—FluorosceinFluorimetry500fmol—Texas redFluorimetry100fmol—RodamineFluorimetry100fmol—IsoluminolChemiluminescence100fmolmetal ionsVoltammetry1fmolbaUrdea M S., Warner B D., Running J A., Stempien M., Clyne J., Horn T., Nucl. Acids Res., 16, 4937 (1988);bIhara T., Maruo Y., Takenaka S., Takagi M., Nucl. Acids Res., 24, 4273 (1996).
At the present stage of development of labeling and detection of oligonucleotide probes, and application of such nucleic acids hybridization technology in research and medical diagnostics, plays an important role in DNA chip technology and its application in genomics (Hoheisel, 1997; Anthony et al., 2001; Lipshutz et al., 1995).
Labelling of DNA-oligonucleotide probes with electrochemical labels and their application in electrochemical detection of nucleic acids was developed only recently. Earlier attempts at electrochemical detection of nucleic acids were based on detection of natural, unlabeled nucleic acid molecules (Wang et al., 1997; Steel et al., 1998; Singhal and Kuhr, 1997). Electrochemical detection of natural nucleic acids is however nonspecific and characterized by high background. Because chemical similarities and similar nucleoside composition of oligonucleotide probes with different base sequences, selective detection of these probes without labelling is very difficult or impossible. In addition, detection of unlabeled oligonucleotide probes is hindered by the electrochemical activity of water. The ferrocene is one of a very few electrochemical labels proposed for labelling of oligonucleotide probes (Ihara et al., 1996; Ihara et al., 1997). In this case however, the label is limited to iron and its redox characteristics.
Electrochemical detection of nucleic acids is disclosed in U.S. Pat. No. 6,391,558 B1 patent dated May 21, 2002. An electrochemical detection system which specifically detects selected nucleic acid segments is described. The system utilizes biological probes such as nucleic acid or peptide nucleic acid probes which are complementary and specifically hybridize with selected nucleic acid segments in order to generate a measurable current when an amperometric potential is applied. The electrochemical signal can be quantified. In the above invention electrochemical detection of only unlabeled nucleic acids using only an amperometric method is proposed.
Nucleic acid detection methods and apparatus, and vessels for detecting nucleic acid are the subject of US 20020064795 A1 patent application dated May 30, 2002. A nucleic acid detection application is disclosed that includes a nucleic acid immobilized electrode constituted by immobilizing a nucleic acid probe to a conductor, a plurality of vessels for bringing the nucleic acid probe into contact with a subject substance, a counter electrode disposed on a bottom surface or a inside surface of the vessel, and an electric circuit for applying a voltage between the nucleic acid immobilized electrode and the counter electrode. A nucleic acid is detected by inserting the nucleic acid immobilized electrode into each vessel containing the subject substance, and using the counter electrode disposed on the bottom surface or inside surface of the vessel to electrically control the reaction.
Quantitative detection of nucleic acids by differential hybridization using electrochemical labelling of samples is described in JP 2002000299 A2 application dated Feb. 8, 2002. A method for quantitative detection of nucleic acids by differential hybridization using immobilized oligonucleotide probes complementary to nucleic acid samples labelled with conductive material, is disclosed. An electric potential is applied and current is measured by differential pulse voltammograms. DNA or PNA (peptide nucleic acid) can be used as probes. Detection of ferrocene labelled oligonucleotides (20mer of adenine, A20) using thymine 20mer probe modified with mercapto hexyl group, or PNA thymine 10 mer modified with 1,2-bis(vinylsulfonyl acetamide) ethane, is described. Hybridization-based gene detection method using intercalator and electrochemical detection of immobilized probe is described in application WO2002/057488 dated Jan. 21, 2002. The gene detection method according to the above invention is characterized in that an intercalator is introduced into the double strand, and the double strand is electrochemically detected. According to the present method, a probe and a sample gene are hybridized in a uniform solution or in a solution in the vicinity of an electrode surface, an intercalator is introduced thereinto for labeling, the probe is then immobilized on the electrode, and the amount of immobilized probe and the amount of double strand are separately detected at the same time, making it possible to accurately detect the amount in which the double strand is produced per unit amount of immobilized probe. An advantage of the present method is that reactions can be conducted with higher efficiency because the hybridization and intercalation are carried out in a solution without immobilizing the probe on the electrode.
Electrochemical detection of nucleic acid hybridization is a subject of U.S. Pat. No. 6,361,951. A method of detecting a nucleic acid (e.g., DNA, RNA) that contains at least one preselected base (e.g., adenine, guanine, 6-mercaptoguanine, 8-oxo-guanine, and 8-oxo-adenine) comprises (a) reacting the nucleic acid with a transition metal complex capable of oxidizing the preselected base in an oxidation-reduction reaction; (b) detecting the oxidation-reduction reaction; and (c) determining the presence or absence of the nucleic acid from the detected oxidation-reduction reaction at the preselected base. The method may be used in a variety of applications, including DNA sequencing, diagnostic assays, and quantitative analysis.
The requirement of the presence of one or more preselected nucleic bases in the detected DNA or RNA is a significant disadvantage of the above method. If the nucleic bases are present in the probe in nature (e.g adenine, guanine) the specificity of the detection is decreased, if they are incorporated into nucleic acids artificially—the additional procedure is required, making the process of detection more complicated. The range of the redox potentials is also limited due to the preselection of nucleic bases, making detection of several probes in the same mixture impossible.
Determination of sequence variations in nucleic acids by electrochemical detection of hybrids using probes labelled with redox groups is described in application WO 2001/007665 A2. The present invention is directed to methods and compounds for the use of self-assembled monolayers to electronically detect nucleic acids, particularly alterations such as nucleotide substitutions (mismatches) and single nucleotide polymorphisms (SNPs). The method uses arrays of probes immobilized in self-assembling monolayers on an electrode surface. Probes and target sequences are labelled with redox groups and hybridization of the probe results in a change in redox potential. Preparation of chips with probes immobilized via disulfide bridges to thiolated DNA is demonstrated and the effects of variables, such as hybridization temperature, are studied. Methods of using competimers, perfectly matching probes that will replace weak or unstable hybrids, to improve the specificity of the hybridization are described.
Electrodes coated with metal complex-containing film and its use for electrochemical detection of nucleic acid bases is described in U.S. Pat. No. 6,180,346 B1. A modified electrode prepared by electropolymerizing a film on the conductive working surface, of an electrode is disclosed. The coated electrode is used for electrochemical detection of nucleic acid bases. The electrode is modified by reductive electropolymerization of a thin film of poly[Ru(vbpy)32+] or poly[Ru(vbpy)32+/vba] (vbpy=4-vinyl-4′ methyl-2,2′-bipyridine and vba=p-vinylbenzoic acid) and the electrode is used for the electrochemical detection of aq. GMP, poly[G], and surface-immobilized single-stranded DNA probes. A DNA probe is attached covalently to the carboxylate group via a carbodiimide reaction followed by amidation of an amino-linked single-stranded DNA. In the presence of these guanine containing moieties, a dramatic enhancement in the oxidative current for the Ru3+/2+ couple (present in the polymeric thin film) due to the catalytic oxidation of guanine is observed. This invention shows example some metal complexes for detection of DNA proves their practical importance.
Electrochemical detection of the hybridization process of a probe and target nucleic with the application of the catalyst of the redox reaction attached to electrode surface is described in the application WO 2001/021635 A2. In the disclosed method DNA- or PNA-oligonucleotide probe containing catalyst of the redox reaction attached to the one end is linked to the electrode surface from the opposite side. Signal is generated as a result of hybridization of the target nucleic acid to the probe and formation of the double stranded structure allowing flow of the electric current from the electrode to the catalyst on the opposite end and initiation of the redox reaction. In this format hybridization processes can be detected using voltametric, amperometric, potentiometric or conductometric methods. Electrochemical detection of the nucleic acid hybridization process with the application of the probes coupled with electrochemical label is a subject of the application WO 2000/031101 A 1. This method can be successfully adapted for the application of electrochemical labelling of a subject of the present invention.
Methods for detecting/quantitating sample nucleic acid fragment by scanning electrochemical microscopy are described in application JP 2001013103 A2 dated Jan. 19, 2001. A highly sensitive method is provided for detecting/quantitating a sample nucleic acid fragment complementary to DNA or PNA (peptide nucleic acid) fragment immobilized on the surface by scanning electrochemical microscopy using DNA or PNA analyzing element. The sample nucleic acid is contacted with the DNA or PNA analyzing element in the presence of a hybrid DNA (or PNA)-binding electrochemical active molecule (e.g, ferrocene-modified intercalate). Then, the sample nucleic acid fragment complementary to the DNA or PNA fragment immobilized on the analyzing element as well as the electrochemical active molecule are bound to the analyzing element. The complementary sample nucleic acid fragment is detected/quantitated by measuring the electric current generated in the electrochemical active molecule-binding region on the analyzing element surface upon applying an electric potential to the analyzing element surface by a scanning electrochemical microscope. This method can be successfully adapted for the application of electrochemical labelling of a subject of the present invention.
An electrochemical method for detecting DNA using DNA sensor and intercalator is described in application JP 2000125865 A2 dated May 9, 2000. The DNA sensor comprises more than two electrodes equipped with the response terminals for output. On the surface of the electrodes, DNA probes possessing the base sequences differing from each other are immobilized. On this DNA sensor, the sample DNA dissociated into single chains are bound with the probe DNA in the presence of the electrochemical activity-embedded-type intercalator, or the intercalator is bound with the DNA hybrid formed beforehand. A DNA gene is detected by measuring the current generated through the intercalator trapped into DNA hybrid. The above method is similar to several methods mentioned earlier utilizing electrochemically labeled intercalating molecule. As a label for the intercalator molecule the electrochemical labels being a subject of the present invention can be also applied. The aim of the present invention is providing new and versatile electrochemical label, a boron cluster, in the form of a metal complex, typically metallacarborane and modified nucleosides or their derivatives comprising thereof. Application of the metallacarborane group is especially desirable because it is characterized by unusually high boron contents, and it can accommodate in its structure a broad variety of other elements, specifically metals, their ions and isotopes. This increases substantially the potential of the proposed new class of molecules beyond their use as electrochemical labels and expand the range of their practical applications. The aims have been unexpectedly achieved in the present invention.